With the NHLBI grant 1R01-HL33142, we have established serially passaged epithelial cell cultures in serum-free medium, from the human trachea. The cells remain sensitive to the trophic effects of vitamin A (retinol) that enhances secretory cell differentiation. High concentrations of Ca++ (1.0-2.0mM), on the other hand, appear to inhibit secretory cell differentiation and stimulate cell proliferation while low concentrations (0.05-0.1 mM) favor secretory cell differentiation and lower rate of proliferation. The major objectives of the renewal application are (1) to further characterize the secreted mucous glycoproteins; (2) prepare monoclonal and polyclonal antibodies to the secreted glycoproteins; (3) identify the types of airway epithelial cells in vivo which synthesize these glycoproteins; (4) identify unique mRNA sequences in vitamin A and Ca++ treated cultures by subtractive hybridization of cDNA libraries; (5) determine whether the rapid effects of Ca++ on differentiation involve (a) effects on plasma membrane and/or alterations in Ca++ transport, (b) alteration in protein phosphorylation, (6) determine if the cell cultures are responsive to the direct effects of neuropharmacological agents, and (7) serially propagate and characterize epithelial cell cultures from the airway of patients with cystic fibrosis. The elucidation of cellular, biochemical and molecular regulations of mucus secretion in normal human tracheal epithelial cell cultures is essential in order to define irregularities in mucus secretion involved in many disease conditions including cystic fibrosis, asthma and inflammation.